Autoantibodies are a characteristic of most autoimmune diseases including rheumatoid arthritis (RA) and appear in the serum many years before the onset of clinical disease, suggesting an early break in B cell tolerance in RA. An important role for B cells in RA has been supported by successful treatment of RA patients with anti-CD20 monoclonal antibodies that eliminate B cells. The underlying mechanisms that account for autoreactive B cell and autoantibody production in RA are poorly defined. We recently analyzed B cell tolerance in untreated active RA patients by testing the specificity of recombinant antibodies cloned from single new emigrant and mature naive B cells. RA patients exhibit defective central and peripheral B cell tolerance checkpoints that result in the accumulation of serf-reactive mature naive B cells, likely contributing to RA pathogenesis. In addition, about half of RA patients showed new emigrant B cells with defective receptor editing, one of the 3 mechanisms that normally ensure B cell tolerance. The other half of the patients displayed new emigrant B cells with unusually long (11 or more amino acids) complementarity determining region 3 (CDRSs) associated to self-reactivity and found enriched in the RA synovium. The long range goal of the proposed research is to determine the mechanisms that regulate B cell tolerance in healthy humans but are defective in RA patients. The working hypothesis is that RA B cells suffer from intrinsic BCR signaling defects that impinge on the proper counterselection of developing autoreactive B cells, and result in the abnormal recruitment and activation of peripheral self-reactive B cells into the synovium. The first aim of the project will consist of characterizing the molecular basis for early defects in B cell tolerance checkpoints in RA by comparing microarray gene expression profiles from control and RA B cell subpopulations (whether or not triggered by their BCR) that may reveal specific BCR signaling defects for the different subgroups of RA patients. The second part of the project will identify alternative B cell tolerance mechanisms such as anergy that can substitute for defective receptor editing in humans. The third part of the project will analyze how B cell tolerance is broken in the synovium of RA patients. These studies have significant implications for understanding how people with autoimmune diseases produce antibodies that attack their body and may provide clues for development of new medications.